Loyola University ChicagoLoyola eCommons

Master's Theses Theses and Dissertations

1977

Experimental Gingivitis in the Albino HamsterJohn Anthony RanieriLoyola University Chicago

This Thesis is brought to you for free and open access by the Theses and Dissertations at Loyola eCommons. It has been accepted for inclusion inMaster's Theses by an authorized administrator of Loyola eCommons. For more information, please contact ecommons@luc.edu.

This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.Copyright © 1977 John Anthony Ranieri

Recommended CitationRanieri, John Anthony, "Experimental Gingivitis in the Albino Hamster" (1977). Master's Theses. Paper 2904.http://ecommons.luc.edu/luc_theses/2904

EXPERIMENTAL GINGIVITIS

IN THE ALBINO HAMSTER

by

John Anthony Ranieri

A Thesis Submitted to the Faculty of th~ Graduate School

of Loyola University of Chicago in Partial Fulfillment

of the Requirements for the Degree of

Master of Science

June

1977

i [.IJBRl\.R"f · · ·····

,OOYOLA UNIVERSIT.X MEDiCA£ CEJ.<U'ER

DEDICATION

To my parents, Louis and Diana Ranieri, whose

encouragement, understanding, and many sacr~fices enabled

me to get here.

ii

ACKNOWLEDGMENTS

I wish to express my deepest appreciation to Dr.

Patrick D. Toto, whose guidance and support made this

thesis possible.

To Dr. Paul Goaz, for his helpful suggestions in

the writing of this thesis.

iii

VITA

John Anthony Ranieri was born on September 30,

1953, in Chicago, Illinois.

He graduat.ed from Holy Cross High School in June

of 1971 and enrolled at Loyola University of Chicago where

he graduated with a Bachelor of Science degree in Biology

in June of 1975.

He was commissioned a Second Lieutenant in the

u.s. Army in June of 1975, and is currently an officer

in the Army Reserve.

He enrolled in the Graduate Program in Oral Biology

at Loyola University School of Dentistry in September of

1975.

iv

TABLE OF CONTENTS

DEDICATION • • • • . . . . . . . . . . Page

ii

ACKNOWLEDGl1ENTS . . . . . . . . . . . . . . . . iii

VITA . . . LIST OF TABLES •

LIST OF FIGURES

CHAPTER

. . . . .

I.

II.

III.

. . . . •. .

INTRODUCTION • • • • • • •

REVIEW OF THE LITERATURE •

l1ATERIALS AND METHODS ••

.RESULTS • • • •

. . .

. . . . . - . . . . . . . . . . .

IV.

v. DISCUSSION • • . . . . . . . . . . . . . VI. SUMMARY AND CONCLUSIONS . . . . . .

BIBLIOGRAPHY • • • • • • • • • •

iv

v

vi

1

3

35

42

57

67

69

,

TABLE

I.

LIST OF TABLES

Studies of Periodontal Disease Using Rodents as the Uodel • • • • • • • •

II. Mean Percentages of Cultivable Organ-

Page

. . . . 6-7

isms in Adult Human Oral Cavity • • • • • • 19

III.

IV.

v.

Composition of the Tes1;. Diet • . • •

Fluorescence of A. naeslundii Reacted with Serum from Experi­mental and Control Hamsters

Agglutination of A. naeslundii with Serum 'from Controland Exper1.mental

. . .

. . . .

Animals . . . . . . . . . . . . . . . . . .

v

36

46

47

LIST OF FIGUP.ES

FIGURE Page

1. Thin smear of Actinomyces naeslundii in experimental serum shm-.ring posl. tJ. ve fluorescence • • • • • • • • • • • • • • • • 43

2. Dense smear of A. naeslundii in experi­mental serum showing posJ.tJ.ve fluores-cence . . . . . . . . . . . . . . . . . . . 4 4

3. ~ naeslundii in control serum showing negative fluorescence • • • • • • • • • 45

4. Bacterial plaque in experimental ham-

5.

6.

7.

sters r • • • • • . . . . . . . . . . Early gingivitis in the hamster . . Early gingivitis in the hamster showing interdental papilla • • .

High magnification of Figure 6 • . .

. . • • 49

• • .- • 50

• • 51

. . . . 52

8. Interdental papilla in an experimental hamster· showing gingivitis • • • • • • • 53

9. Interdental papilla from a control hamster • • • • • • • • • • • • • • • • 54

10.

11.

High magnification of Figure 9

Low magnification of Figure 9

vi

·. . . . • • • 55

• • • • 56

CHAPTER I

INTRODUCTION

Chronic gingivitis is an inflammatory disorder of

the gingiva. Past investigations have demonstrated that

microbial plaque lying adjacent to the host's gingival tis­

sues is the primary etiologic factor, initiating gingival

inflammation and the ensuing periodontal disease (Loe,

Theilade, and Jensen, 1965}.

The bacteria and their products cause gingivitis by

stimulating a reaction in the host's immune system. The

humoral immune response in the host produces specific anti­

body as a result of the invasion by bacterial products act­

ing as antigens (Nisengard and Beutner, 1970a, Gilmour and

Nisengard, 19731.

Numerous studies have been conducted implicating

Actinomyces naeslundii as a primary etiologic agent in gin­

givitis and periodontitis (Socransky, Hubersak, and Propas,

1970, Marttala, Toto, and Gargiulo, 1974). The antigenic

activity of cell portions of A. naeslundii has been demon­

strated (Irving, Heeley, and Socransky, 1975).

In addition, specific antibody activity against

Actinomyces naeslundii, ~ israelii, ~ viscosus, and

1

2

streptococcus mutans has been demonstrated in the gingiva

of patients with chronic periodontal disease by use of the

fluorescent antibody technique (Marttala, Toto, and Gargiulo,

19 74) •

The purpose of this investigation is to demonstrate

an induction of specific antibody activity against ~ ~­

lundii in albino hamsters resulting from an oral exposure

to this microorganism experimentally. The role of A. naes-

lundii in the initiation of experimental gingivitis in

these animals will also be investigated.

CHAPTER II

REVIEW OF THE LITERATURE

Periodontal disease has been defined as the morbid

response o£ the periodontal tissues to extrinsic local

irritants. This disease has afflicted man probably since

the beginning of time and is one of the two major oral

diseases. Periodontal disease is a major health problem

and is said to be the most common cause of tooth loss,

affecting millions in the United States alone. Further­

more, periodontal disease may become an even greater threat

in the future, since longevity is increasing (Rizzo, 1973).

Much of the research concerned with periodontal dis­

ease has depended on the use of experimental animals.

For over twenty years rodents have been used as mod­

els for periodontal research. The early observations of

Jordan and Keyes (1964} described a condition in hamsters

characterized by gingival accumulation'of plaque-like ma­

terial around the molar teeth. Resultant soft tissue

disturbances and alveolar bone loss indicated the potential

of the hamster for studies on periodontal disease. Dental

plaques have been defined as " ••• dense, non-calcified

3

4

~acterial masses so.firmly adherent to the tooth surface

that they resist wash off by salivary flow." (Gibbons and

van Haute, 1973).

Primary consideration in the choice of an animal

model must be given to the complexity of the etiological

forces involved in the disease under investigation. A

simple model may be adequate where the etiological effect

is potent or easily manipulated. If, however, the etio­

logical effect is dependent upon a multiplicity of con­

ditions, as in periodontal disease, the results might vary

with different animal species. In order to study all the

aspects of the disease, various spec'ies may be needed for

each (Frenkel, 1969).

The way in which the model differs from the original

is governed by fidelity and discrimination. Fidelity refers

to the overall differences between the model and the orig­

inal while discrimination describes the ability of the

model to reproduce a particular property of the original.

There may be drawbacks to using a model of high fidelity:

this makes it impossible to simplify a complex disease

situation to the stage at which elements of importance can

be recognized and evaluated. Further, high fidelity alone

may not justify the added difficulties presented. The

rationale of using a higher animal to investigate human

5

disease concepts should be related to some particular fea­

ture that is absent or unsuitable for some reason in the

lower animal. Frenkel (1969) has stated that, to be mean­

ingful, the process under investigation must be analogous

to that occurring in man.

The choice of a model for periodontal disease re­

search depends also upon the questions posed. This research

deals with the role of Actinomyces naeslundii in the etiol­

ogy of gingivitis in the albino hamster. In view of the

fact that this is. a restricted phase of the. disease, the

model chosen should show good discrimination for this phase.

Over the years periodontal resear9hers have used

various species of experimental animals. These include the

Beagle dog (Saxe, Greene, Bohannon, and Vermillion, 1967),

the Syrian hamster (Keyes and Fitzgerald, 1970), the monkey

(Krygier, .Genco, Mashino, and Hausmann, 1973), and the Mon­

golia! gerbil (Moskow, Wasserman, and Rennert, 1968), among

others. The most widely used model has been the rodent

(See Table I) •

The utility of rodents has been demonstrated by

their widespread use in periodontal disease research. Ro­

dents have advantages in small size, relatively low cost of

upkeep, and ease in handling, breeding, and housing (Jordan,

1971). The rodent is a predictable model in the sense of

TABLE I

Studies of Periodontal Disease Using Rodents as the Hodel

INVESTIGATOR EXPERI- TEST ORGANISM .lWi'tAIL

t1itchell & Chernausek

1951

Gupta, Ausk?ps, & Shaw

1957 1968

Jordan & Keyes 1964

Keyes & Jordan

1964

ANI HAL

Syrian hamster

Rice rat

Albino hamster

Syrian hamster

None introduced

None introduced

' Streptococci······ Sarcina··········· G- rods··········· G+ aerobic, fil-

amentous bacteria

Plaque organisms from perio. disease­prone golden & cream hamsters

DIET USED

Finely ground, high carbohydrate, with ammoniated products················

\-Ji thout ammon. products················

#700 (Shaw, 194 7) •••••••• #700 with antibiotics····

#2000, 56% sucrose······· #2000, 56% sucrose······· tf2000, 56% sucrose· • • • • • •

#2000, 56% sucrose·······

IF ANY DISEASE

Periodontal disease

"a reduced am't of perio.involvement"

Perio. disease Major reduction

in perio. dis.

None observed None observed None observed

Perio. disease includ. bone resorption

#2000·············· Perio. disease

INVESTIGATOR

Dick & Shaw, 1966

Socransky, Hubersak, & Propas,

1970

Jordan, Keyes, & Bel lack

1972'

f.1:iller & Ripley 1975

EXPERI­MENTAL ANI.t-'IAL

Rice rat

Sprague­Dawley rats

Albino hamster & gnotobiotic rats

Syrian hamster

TABLE I {cont.)

TEST ORGANISM

Organisms in feces of rice rats with fully developed period. lesions

Actinomyces naeslundii

Actinomyces of l).uman origin

Actinomyces viscosus

DIET USED

#700 {Shaw, 1947)

#2000 {Keyes & Jordan, 1964)

#2000

#2000 {Keyes & Jordan, 1964).

IF ANY DISEASE

Periodontal disease

Periodontal disease, alveolar bone destruc­tion, subgin. plaque formation, hair irnpact­ation, root caries

Gingival distortion, pocketing, alveolo­clasia, root caries

Inflammation, plaque formation, bone resorption

reproducing the disease with a high degree of reliability

{Keyes, 1964, Dick and Shaw, 1966).

8

Dietary studies which have been performed on ro­

dents are in agreement with observations in human popula­

tions (Jordan, Keyes, and Lim, 1969). Starch containing

diets support the infection of Actinomyces viscosus in ani­

mals (Keyes and Jordan, 1964) • It has been found that, in

human populations, those that consume starch as the princi­

ple carbohydrate and take in little refined sugar are likely

to develop periodontal disease rather than caries (Jordan,

1971).

Observations have shown that the gingival plaque

found in rodents is similar to that which invests the cervi­

cal regions of the teeth in humans and it has been demon­

strated that both contain species of Actinomyces (Jordan

and Keyes, 1964, Slack, Lanfried, and Gerencser, 1971).

Cervicoradicular infections have been induced in rodents by

the use of isolates of Actinomycete-like bacteria from human

plaque (Jordan and Keyes, 1964).

The implantation of Odontomyces viscosus in albino

hamsters is supported by sucrose or starch diets. Glucose

and fructose also support 0. viscosus implantation in gold­

en hamsters (Jordan, Keyes, and Lim, 1969).

9

The use of. germfree (gnotobiotic) rodents has proven

extremely useful in testing the pathogenic properties of

specific bacterial species. This has been done by observ­

ing any pathological changes which may occur in these ani­

mals subsequent to monoinfection with a particular bacterial

specie(s} (Socransky, Hubersak, and Propas, 1970, Jordan,

Keyes, and Bellack, 1972).

The initiating role of bacteria in inflammatory

gingival disease has been studied at length, especially

over the last decade (Lee, Theilade, and Jensen, 1965,

Socransky, Hubersak, and Propas, 1970, Jordan, Keyes, and

Be1lack, 1972, among others}.

Rovin, Costich, and Gordan (1966}, in their work

with germfree rats, demonstrated periodontal inflammation

could not occur in the absence of microorganisms. They ob­

served both local irritation and bacteria were necessary to

produce periodontal inflammation in their model.

Bacteria or their products may' directly damage perio­

dontal tissues, however, it is likely that the inflammatory

process of periodontal disease is a result of the inter­

action between bacterial products and the host's immune

system (Bahn, 1970).

In the oral cavity, a delicate balance exists be­

tween the defense mechanisms of the host and the constant

10

challenge posed by the oral flora and its products. An up­

set in this balance may be caused by factors such as physi­

cal irritants including calculus, as well as trauma and sys­

temic disease. Such an upset may lead to the inflammatory

process of periodontal disease (Bahn, 1970}.

The immune system, composed of humoral and cellular

mechanisms, has as a primary functiqn the protection of the

host against microbial invasion. When antibodies (humoral)

or lymphocytes (cellular} react with specific antigens,

these reactions may trigger localized~~mmediate and delayed

hypersensitivities respectively, resulting in inflammation.

A local inflammatory response may serve as the main defense

against microbial invasion. If it persists, however, it

may ca~se destructiqn of periodonta~ tissues (Snyderman,

1973). The type of immunologic response induced is a result

of the host's initial contact with the antigen (Baram and

Arnold, 1970) •

Agents which include antigen-antibody complexes,

bacterial endotoxin, and some.proteases are capable of ac­

tivating the complement sequence. As a result, several as­

pects of the inflammatory process, vasodilation, vascular

permeability, smooth muscle constriction, and chemotaxis

for leukocytes, are mediated by biologically active pep­

tides released by the complement components C3a and CSa,

11

which are anaphylatoxins (Snyderman, Phillips, and Mergen­

hagen, 1971). These mediators include histamine, serotonin,

and bradykinin.

Jensen, Snyderman, and Mergenhagen (1969) found

that C5a injected intradermally in. guinea pigs resulted in

localized enhanced vascular permeability, vasodilation, and

a massive buildup of polymorphonuylear leukocytes. Further,

the ability of C5a to contract smooth muscle in vitro has

been demonstrated (Jensen, 1967).

As a consequence of an induction of inflammation by

pharmacologically active mediators, there is increased exu­

date from regional capillaries into tissues causing edema

and increased penetration of bacterial antigens. Bacteria

may continue to stimulate antibody formation locally in

regional lymph nodes and in adjacent tissues (Mergenhagen,

1967).

The main source of histamine in the connective tis­

sues is the mast cells (Seyle, 1965). Complement has been

implicated in the formation of a factor that releases this

histamine from the mast cells (Hook, Snyderman, and Mergen­

hagen, 1970). Several biological properties of heparin, also

released from mast cells, are related to the inflammatory

process, but these are less significant than its ability to

enhance bone resorption in vitro (Goldhaber, 1965).

The gingiva has been revealed to be a potential

reservoir of histamine, the major portion of which is

stored in the mast cells (Schwartz and Dibblee, l975b).

When appropriately challenged in vitro, the gingiva can . . .

12

release this histamine. The authors reported a signifi­

cant release of gingival histamine subsequent to challenge

by each of the enzymes protease, collagenase, trypsin, and

chymotrypsin. · This histamine release may contribute to

chronic periodontal inflammation.

IgE immunoglobulins are cytotropic and are known

to have a strong affinity for the surface receptors of mast

cells of homologous species (Papermaster, 1972). These

findings have been supported by the recent investigation

of Schwartz and Dibblee (1975a} who demonstrated a close

association of IgE with gingival mast cell histamine re­

lease in vitro.

Bacterial antigens may be categorized into three

main types. These are: 1. the peptidoglycans, 2. lipo-

polysaccharide endotoxins, and 3. proteins such as enzymes,

structural proteins, and protein complexes containing poly­

saccharide and lipid, which are produced by oral micro­

organisms (Bahn, 1970). These may be allergens, causing

an allergic or hypersensitive response in some predisposed

individuals. Chronic inflammation may result from the

13

continued presence of these allergens in plaque (Bahn, 1970).

Peptidoglycans comprise the muco-peptide layer of the cell

wall of gram positive bacteria. Peptidoglycan is immuno­

genic and, combined with complement present in gingival

fluid, may initiate an inflammatory response (Bahn, 1970) •

Penetration of the gingival epithelium and subse­

.quent interaction with local antibodies by the. gingival sul-

cus bacteria Streptococcus mitis, Streptococcus salivarius,

and Neisseria catarrhalis has been demonstrated (Mayron, 1973).

Polymorphonuclear leukocytes (PMN's} are phagocytic

cells which may contribute to tissue destruction-by releas­

ing intracellular products from their lysosomes during

phagocytosis (Cohn and Hirsch, 1960, Cochrane, 1969). In-

deed, a widening of the intracellular spaces in human gin-

gival sulcus epithelium subsequent to the application of

intracellular enzymes from human PMN's has been observed

(Thilander, 1963}. Further, it has been demonstrated that

the early inflammatory response to intradermal injections

of mixed cultures of oral bacteria (Jensen and Mergenhagen,

1964) and to intradermal injection of dental plaque contain­

ing viable bacteria (Taichrnan, Friedman, and Uriukara, 1966)

is greatly reduced in rabbits previously rendered leukopenic.

Significantly, a prevention of tissue damage due to the in­

flammatory response in the Arthus and local Schwartzman

14

reactions has been observed in leukopenic rabbits (Stetson,

1951). In addition to these effects, it has been demon-

strated that lysosomal enzymes derived from inflammatory

cells are capable of stimulating bone decalcification in

vitro (Fell, Coombs, and Dingle, 1966}.

It has been found that the number of PMN's in the

gingival sulcus during the course of,experimental gingivi­

tis in man is increased (Lee, Theilade, and Jensen, 1965) •

Further, it has been postulated that the number of PMN's

present may reflect the degree of inflammation in the_ gingi­

val tissues (Klinkhamer, 1968).

Experimentally, in vitro and in vivo, it has been

demonstrated that PMN's respond to chemotactic factors

(Harris, 1954, Keller and Sorkin, 1967).

Tempel, Snyderman, Jordan, and Mergenhagen (1970)

classified agents causing chemotaxis for PMN's into two

categories: agents which exert their influence on PMN's

directly, such as bacterial chemotactic factors, and those

agents which are chemotactic by virtue of an interaction

with host factors. The authors, in their investigation of

factors chemotactic for PMN's, found that all strains of

oral bacteria evaluated produced chemotaxis for PMN's.

That viable bacteria produce a product directly chemotactic

for neutrophils in vitro had previously been documented

15

(Keller and Sorkin, 1967, Ward, Lepow, and Newman, 1968, ·

Walker, Barlet, and Kurtz, 1969}. This work was supported

by Snyderman (1973} who observed filtrates from cultures

of three strains of bacteria isolated from human dental

plaque to be distinctly chemotactic for PMN's.

The recent investigation of Miller, Folke, and

Umana (1975} demonstrated dental plaque to be consistently

chemotactic in vitro for human PMN's. Plaque supernatant

was found to be approximately sixty percent less chemotac­

tic than whole plaque, implicating the bacteria as primary

chemotactic factors. This contention was substantiated by

the demonstration of negligible chemotactic activity in the

ultrafiltrate, which contained no bacteria. The authors

found antigen-antibody complexes, used as positive controls,

were more chemotactic than whole plaque·suspensions. This

finding supported the work of Boyden (19621 who had previ­

ously demonstrated the chemotactic activity of ag-ab complex-

es.

In addition to the humoral mechanism of the immune

system and its role in the etiology of periodontal disease,

the cellular response has been studied at length (Burnett

and Scherp, 1968, Bahn, 1970, Irving, Heeley, and Socransky,

1975, among others}. An analysis of the cellular response

in periodontal patients has shown that these patients have

16

lymphocytes which are more reactive than those found in

normal individuals (Horton, Leikin, and Oppenheim, 1972).

Further, it has been demonstrated that lymphocytes from

individuals with cellular immune hypersensitivity undergo

a proliferative response (lymphocyte transformation} when

cultured in vitro with a specific antigen (Oppenheim, 1969).

Significantly, it has been observed that lymphocytes re­

lease soluble mediators of inflammation before and during

transformation (Pick and Turk, 1972). Included among these

lymphocyte-derived mediators are a monocyte chemotactic

factor (LDCF), a cytotoxic factor (Lymphotoxin},-and a

macrophage inhibitory factor (MIF). These mediators are

called lymphokines (Snyderman, 1973).

Pertinent at this point in the discussion is the

observation that lymphocytes obtained from patients with

destructive periodontal disease (as evidenced by a high

Periodontal Index or PDI) are distinctly cytotoxic for

gingival epithelial cells in vitro. At the same time, no

significant lymphocytotoxicity was demonstrated on the part

of aggressor lymphocytes from periodontal disease-free (low

PDI} patients (Movius, Rogers III, and Reeve, 1975}.

Other factors reportedly released by stimulated

lymphocytes include mitogenic factors (Dumonde, Howson, and

Wolstencroft, 1967} and a factor that inhibits cellular

17

proliferation (Green, Cooperband, Rutstein, and Kibrick,

1970), the latter effect possibly preventing tissue repair.

Studies have demonstrated substances in plaque,

derived from oral bacteria, are capable of initiating a

cellular immune response. Ivanyi and Lehner (1970) were

successful in isolating a lymphoproliferative initiator

material from four strains of bacteria commonly found in

the human oral cavity. It has been found that lymphocytes

from patients having periodontal qisease undergo a prolif­

erative response when exposed to plaque antigens. Further,

the degree of lymphocyte transformation is directly related

to the severity of the lymphocyte donor's periodontal dis­

ease (Horton, Leikin, and Oppenheim, 1972).

As a result of the interaction of plaque with leu­

kocytes cultured from periodontal patients, a release of

lyrnphotoxin has been observed (Horton, Oppenheim, and

Mergenhagen, 1973). In addition, the authors found that

the amount of lyrnphotoxin produced was directly proportional

to the severity of the leukocyte donor's periodontal dis­

ease. The lymphotoxin was cytotoxic for human fibroblast

cultures in vitro.

Supernatant fluids of human leukocyte cultures

stimulated by dental extracts have been found to contain,

in addition to lymphotoxin, a substance which has produced

18

bone resorption in organ cultures of fetal rat bone. This

effect may have been a consequence of an observed increase

in the number of active osteoclasts in the bone cultures,

apparently caused by this substance. As an osteoclast acti-

vating factor (OAF}, this lyrnphokine may be the cause of the

alveolar bone loss seen in advanced periodontal disease

(Horton, Raisz, Simmons, Oppenheim, and Mergenhagen, 1972).

The presence of microorganisms in the gingival crev-

ice has been known since their description by Leeuwenhoek in

1678 (Arnim, 1967). A review of the literature reveals that

periodontal inflammation does not occur in the absence of

microorganisms. For example, in an investigation of perio-

dontal disease in rats, Rovin, Costich, and Gordon (1966)

observed that, in germfree rats, local irritation in the

form of silk ligatures placed at the cervical margins of the

first molars was not sufficient to cause periodontal inflam-

mation. Inflammation occurred only in the presence of .

microorganisms found in conventional rats.

The observation has been made that periodontal dis-

ease is infectious in origin and has not been known to occur

in the absence of an infectious agent (Ellison, 1970). A

table of the normal human oral flora is found in Table II.

The accumulation of bacterial plaque in man has been

directly correlated with increased inflammation of gingival

TABLE II

1-tean Percentages of Cultivable Organisms in Adult HummOral Cavity (Socransky and Manganiello, 1971)

Genus or Type Gingival Crevice Dental Ton que Saliva Area Plaque

Gram + Facultative Cocci 28.8 28.2 44.8 46.2 Streptococci 27.1 27.9 38.3 41.0 s. salivarius N.D. N.D. 8.2 4.6 Staphylococci 1.7 0.3 6.5 4.0

Enterococci 7.2 N.D. 1.3 Gram + Anaerobic Cocci 7.4 12.6 4.2 13.0 Gram - Facultative Cocci 0.4 0.4 3.4 1.2 Gram - Anaerobic Cocci 10.7 6.4 16.0 15.9 Gram + Facultative Rods 15.3 23.8 13.0 11.8 Gram + Anaerobic Rods 20.2 18.4 8.2 4.8 Gram - Facultative Rods 1.2 N.D. 3.2 2.3 Gram - 1illaerobic Rods- 16.1 10.4 8.2 4.8

Fusobacterium 1.9 4.1 0.7 0.3 Bacteroides melanin-

ogenicus 4.7 H.D. 0.2 N.D. Vibrio sputorum 3. 8 .. 1.3 2.2 2.1 Other Bacteroides 5.6 4.8 5.1 2.4

Spirochetes 1.0 N.D. N.D. N.D.

N.D.= not detected

20

tissue in healthy individuais (Loe, Theilade, and Jensen,

1965). In this study, withdrawal of oral hygiene measures

in twelve human patients originally having clinically nor­

mal gingivae led to the accumulation of soft debris and de­

velopment of marginal gingivitis in all subjects. Concur­

rent bacteriological examinations demonstrated an increase

r:···.· .• ·.·. in the number of microorganisms in · the gingival area and

distinct changes in the relative composition of the flora.

Reinstitution of oral hygiene resulted in healthy gingival

f

~ ~

conditions and the reestablishment ofithe original flora.

While these results do not, by themselves, prove

the initiating role of bacteria in periodontal disease,

The fact that . • • the change in microflora occurred before gingivitis was clinically diagnosed may indicate that the microorganisms play a role in the initiation of periodontal inflammation. (Lt:>e, et al., 1965).

Similar findings relating to ah increase in the total num-

bers of organisms and a relative increase in specific organ-

isms in patients having non-specific gingivitis versus pa­

tients with normal gingivae have also been reported (Schultz­

Haudt, Bruce, and Bibby, 1953).

Collagen breakdown by certain enzymes from the gin-

gival bacteria, Bacteroides melaninogenicus has been reported

(Macdonald, Gibbons, and Socransky, 1960). Further, it has

been demonstrated that hyaluronidase applied locally in the

21

gingival sulcus causes a widening of the intercellular

spaces of the epithelium (Schultz-Haudt, Dewar, and Bibby,

1963) •

Much research has been done concerning the role of

bacterial plaque as an essential factor in the initiation

of periodontal inflammation. Plaque components have been

demonstrated to be nutrients for bacterial growth (Wilder­

man, 1962). In a study of periodontal disease in dogs, Gad

(1968} observed signs of the disease in ninety-seven percent

of the dogs, as well as calculus and debris.

A positive correlation between the amount of soft

dental deposits and the severity of gingivitis has been dem­

onstrated in the beagle dog (Saxe, Greene, Bohannon, and

Vermillion, 1967}. The authors found that the etiologic

factors in periodontal disease seem to be identical in hu­

mans and dogs. In further support of a correlation between

bacterial plaque and periodontal disease was the demonstra­

tion that a gingivitis which gradually develops into perio­

dontitis can be induced in young beagle dogs simply by al­

lowing plaque to accumulate on the teeth (Lindhe, Hamp, and

Lee, 1973}. The authors observed three stages in the dis­

ease process: 1. Subclinical gingivitis, 2. Clinical gingi­

vitis, 3. Periodontal breakdown.

22

The development of a periodontal pathosis associated

with the impactation of food and other debris and calculus

and plaque has been reported in golden hamsters which had

been sustained on a high starch diet (King and Gimson, 1948-

49). In addition, a slowly progressing, insidious inflam­

matory periodontal breakdown apparently related to the ac-

cumulation of plaque and calculus-like accretions has been

observed in the Mongolian gerbil. Of relevance to man was

the demonstration of many morphological similarities in the

periodontal lesion in the two species (Moskow, Wasserman,

and Rennert, 1968) •

An effective tool in the investigation of the pos­

sible roles of specific types of bacteria in peri~dontal

disease has been the antibiotics. These chemotherapeutic

agents have been used in the selective elimination from the

oral flora of certain bac.terial species. Subsequent changes

observed in the oral health of the host may suggest a rela-

tionship between the type of bacteria which had been elim-

inated and the changes observed. It has been reported that

the administration of either penicillin or streptomycin in

the diet of rice rats with periodontal disease resulted in

major reductions in the disease in these animals (Gupta,

Auskaps, and Shaw, 1957}.

23

Shaw, Griffiths, and Auskaps, (1961), evaluated the

role of antibiotics in periodontal therapy in the rice rat.

Two experiments were designed, one to determine the curative

ability and the other the preventive ability of four anti­

biotics administered as a dietary supplement. In experiment

one, which was preventive, both high and low concentrations

of Erythromycin glucoheptonate resulted in significant pre­

vention of lesions of soft and hard tissues. Two concentra­

tions of Polymixin B sulfate resulted in striking prevention

of soft tissue lesions and lesser prevention in hard tissue

lesions. In experiment two, which was curative, the animals

were maintained for ten weeks on high carbohydrate diet 700

followed by ten weeks of antibiotic supplementation with

Benzylpenicillin plus Streptomycin sulfate and Benzylpeni­

cillin plus Oxtytetracycline hydrochloride.

Antibiotic regimens were able to stop the progress

of lesions of the calcified tissues but were not adequate

to assist in any repair, as they failed to cause regression

of bone tissue lesions. In soft tissues they stopped the

progress of inflammation and allowed the lesions to heal.

At low levels (.005% - .01%) Benzylpenicillin and Erythro­

mycin glucoheptonate were effective in prevention while

Polymixin B proved ineffective in prevention at these same

low levels. These studies indicate a strong microbial

24

component in the etiology of the periodontal syndrome of the

rice rat.

It has been demonstrated that the microorganisms

responsible for the periodontal syndrome in the rice rat are

readily transmitted between individuals (Dick and Shaw,

1966}. The authors obtained results in agreement with those

of Gupta et al. {1957} with respect to antibiotic therapy.

It was found that rice rats prone to develop periodontal

disease when given high carbohydrate diet 700 were rendered

relatively inactive with respect to uhis disease when sub­

jected to a period of penicillin supplementation-. After

cessation of penicillin administration, inoculation of the

oral cavity with organisms from the feces of rats having

fully developed periodontal lesions resulted in rapid ini­

tiation and progression of the destructive process. Compa­

rable oral inoculation with fecal bacteria of rice rats of

the strain which does not develop the disease readily re­

sulted in a greatly increased rate of initiation and pro­

gression of lesions. As little as one inoculation on the

first day after penicillin cessation was sufficient to re­

sult in a return to the same rapid rate of development of

the disease as when there had been no suppression of the

flora by penicillin. These results, implicating entero­

cocci in the initiation of periodontal disease in the rice

25

rat, supported the findings of Macdonald, Socransky, and

Sawyer (1960}, and Socransky, Macdonald, Sawyer, and Aus-

kaps (1960} •

The antibiotic vancomycin, an agent which affects

gram-positive bacteria, has been utilized in a study of il experimental gingivitis in man (Jensen, Loe, Schiott, and

Theilade, 1968}. The authors subjected an experimental

. group to an oral vancomycin rinse for a period of three

weeks, assuming this would result in the absence of gram-

positive bacteria·from the oral flora.- Both control and

experimental groups abstained from any oral hygiene meas-

ures during the experimental period. Gingivitis developed

in all individuals in the experimental group, implying that

the gram-negative flora is able to form plaque and elicit

gingivitis in man.

Numerous studies have been conducted implicating

various strains of Actinomyces as primary etiologic agents

in gingivitis and periodontitis. Keyes and Jordan {1964)

demonstrated filamentous bacteria to be an infectious and

'transmissible component. in the causation of periodontal le-

sions in the Syrian hamster. Qf the three strains of ham-

ster produced by the. NIH Animal Production Center, the gold-

en or Syrian, the cream, and the albino, an active type of

periodontal disease develops readily in the golden and the

26

cream when they are administered a high carbohydrate diet

but fails to develop in the albino under similar conditions.

In addition, Actinomyces species are found in negligible

numbers in the albino hamster. In their research, however,

Keyes and Jordan demonstrated that the disease can be in­

duced in non-infected albino animals by inoculation of

scrapings of subgingival plaque obtained from infected ani­

mals. The disease is then transmitted from generation to

generation. Filamentous Actinomyces were found in high

numbers in cultures of the plaque material. Significantly,

the disease did not develop in uninoculated controls. In

a "Generation Study" group, passage of the disease from

mother to offspring by coprophagy was investigated. Passage

of the disease was terminated by the use of penicillin.

In a related study of_ gram-positive filamentous bac­

teria as etiologic agents of experimental periodontal dis­

ease in hamsters, Jordan and Keyes (1964} utilized pure cul­

tures of these organisms obtained from the plaque of in­

fected golden and cream hamsters. These cultures were cap­

able of inducing the disease when inoculated orally into un­

infected albino hamsters. Other plaque organisms, including

streptococci and unidentified gram-negative rods, failed to

induce the disease subsequent to similar inoculation of un­

infected animals. The periodontal syndrome induced by the

27

Actinomyces closely resembled that of the natural infection.

The authors concluded that filamentous organisms can be an

essential bacterial component in the etiology of periodon­

tal disease in their model.

This conclusion was supported by Socransky, Hubersak,

and Propas (1970} who suggested that,

• • • certain strains of Actinomyces resident in the human gingival crevice are capable of colonizing the cervical and root surfaces of teeth, of forming destructive bacterial plaques that lead to pocket formation, destruction of alveolar bone, root caries, and exfoliation.

The authors isolated and then cultured 122 bacterial strains

from human gingival debris. Nineteen of these formed large

amounts of plaque on all test media and proved to be gram-

positive rods. One strain, Actinomyces naeslundii, was then

used in the monoinfection of gnotobiotic Sprague Dawley

rats. Four experiments were conducted, the age of the ani-

mals at sacrifice ranging from 90 to 455 days. In all

cases, experimental animals revealed severe destruction of

the alveolar bone associated with subgingival plaque forma-

tion, hair impactation, and root caries. Bone loss incre-

ment was determined in each experiment. In each case, ani-

mals monoinfected with Actinomyces naeslundii demonstrated

significantly greater bone loss versus control animals.

28

Similar results were obtained by Jordan, Keyes, and

Bellack (1972) who inoculated the oral cavities of albino

hamsters and gnotobiotic rats with species of Actinomyces

of human origin. The gnotobiotic rats, after infection,

showed gingival distortion, pocketing, alveoloclasia, and

root caries associated with impactation of hair fibers and

formation of bacterial mats on root surfaces. The denti-

tion was often destroyed by an invasion of these microorgan­

isms which, in some cases, penetrated to the ~ulp chamber.

Beveridge and Goldner {1973} sought a statistical -

relationship between the presence of human subgingival

anaerobic diphtheroids and periodontal disease. As a result

of assays in periodontal and non-periodontal patients, the

amount of anaerobic diphtheroids in the subgingival plaque

was determined. The authors found a fifty-nine percent

incidence of these organisms in the periodontally diseased

patients while the clinically normal individuals showed

only four percent.

In a recent assay of human subgingival microflora,

it was found that a significantly greater number of Actino-

myces was present in the flora of periodontal patients as

compared to control subjects (Williams, Pantalone, and

Sherris, 1976).

29

In an investigation of a limited number of gram­

negative bacteria in the healthy and inflamed human gingi­

val crevice, Helderman (1976} observed a significant in­

crease in the proportion of cultivable gram-negative organ­

isms with increasing gingival index score. The organisms

implicated were Vibrio sputorum and Fusobacterium nucleatum.

These 'findings seem to refute the previous work of Darwish,

Hyyppa, Manganiello, and Socransky (19731 who detected no

gram-negative bacteria in a study of the predominant cul­

tivableJrnicroorganisms in early:periodontitis supra and

subgingivally. This difference may be reconciled in view

of the fact that each study dealt with a different disease

state. The former authors obtained.data from patients dem­

onstrating gingival inflammation while the latter study

dealt with a disease state which included slight bone loss,

although both groups had_ gingivitis.

In the investigation of the presence of specific

antigens or antibodies in certain disease states, agglutin­

ation of particulate antigens has been described as a sen­

sitive analytical method. The agglutination test was orig­

inally used in the detection of "agglutinin," or antibodies

produced as a result of injection of cells or bacteria.

Very small quantities of antibody can be detected by this

method since only a small number of bivalent antibody mole-

30

cules is necessary for the formation of the antigen-antibody

lattice (Hauro~itz, 1968}. The a~glutination of bacterial

cells has been investigated quantitatively (Heidelberger

and Kabat, 1937} •

Antigen-antibody complexes were made visible by the

labelling of the antibody molecules with fluorescent com-

pounds in 1956 by Moody, Goldman, and Thomason and Thomason,

Moody, and Goldman.

Antigen-antibody reactions which are not always

demonstrable by agglutination or other types of serological

tests can often by observed by the use of fluorescent anti­

body techniques (Cherry, Goldman, and Carski, 1960}. F.A.

techniques have been used in microbiology since the work of

Coons in 1942. The value of the F.A. technique as a highly

specific method in the identification of bacterial antigens

has been demonstrated since the work of Goldman (1953, 1954}.

The use of fluorescein has been widely indicated

for use as a label because its yellow-green color is one to

which the eye is most sensitive and in normal tissue is

rarely encountered as autofluorescence. In addition, fluo-

rescein is highly efficient (Cherry, Goldman, and Carski,

1960). The isothiocyanate derivative of fluorescein has

been used in the labelling of globulins since 1958 (Mar-

shall, Eveland, and Smith, 1958, Riggs, Seiwald, Burck-

31

halter, Downs, and Metcalf, 1958}.

Actinomyces naeslundii is a gram-positive, pleomor­

phic, branching, anaerobic rod (Socransky, Hubersak, and

Propas, 1970) which is a normal inhabitant of the human oral

cavity (Williams,.Pantalone, and Sherris, 1976).

Labelled antiglobulins conjugated with FITC were suc­

cessfully used in the identification of Actinomyces naeslun­

dii and A. israelii by Lambert, Brown, and Georg in 1967.

A. naeslundii was found to be serologically distinct from

morphologically similar A. israelii. Comparison of F.A.

results with those of the agar~gel diffusion test, which

.was also employed, indicated that the two tests are about

equal in sensitivity, but the F.A. test was more specific,

since several cross-reactions were encountered with hetero­

logous species in the agar-gel diffusion test, while the

F.A. test showed no cross reactions. This serologic char­

acterization of A. naeslundii was supported by Socransky,

Hubersak, and Propas in 1970.

The fluorescent antibody technique has been used to

identify species of Actinomyces, including A. naeslundii,

in human dental plaque by Snyder, Bullock, and Parker {1967)~

Slack, Mance, and Gerencser (1973) demonstrated that direct

F.A. technique can be employed to specifically identify A.

naeslundii, among other species in early (3 -days) and late

32

(80 days} plaque.

The further investigation of Blank and Georg (1968)

indicated that the fluorescent antibody technique provided

rapid and specific identification of several Actinomyces

species, including ~ naeslundii, both in clinical material

and isolated in pure culture.

A. naeslundii has been identified in human dental

calculus by the fluorescent antibody technique (Slack, Lan-

fried, and Gerencser, 1971}. In addition,~ naeslundii,

as well as A. viscosus, has been identified from periodontal

pockets (Cummings and Hageage, 1974}.

Marttala, Toto, and Gargiulo, (1974) have demon-

strated specific antibody activity against A. naeslundii,

~ israelii, ~ visco·sus, and Streptococcus mitans in the

. gingiva of patients with chronic periodontal disease.

That specific serum antibodies are produced in pa-

tients with periodontal disease has been documented (Nisen-.

gard and Beutner, 1970, Gilmour and Nisengard, 1973, Cum-

mings and Hageage, 1974). Experimental ingestion of bacte-

ria in gnotobiotic rats is known to induce specific anti-

body production in these animals (Michalek, McGhee, Mestecky,

Arnold, and Bozzo, 1976).

Both immediate hypersensitivity and the presence of

serum antibodies in humans to Actinomyces has been documented

33

(Nisengard and Beutner, 197Q a & b}. In addition, the au­

thors found that the percentage of allergic individuals in­

creased with increased severity of periodontal inflammation.

Significantly, antibody levels in humans to A. naeslundii and

~ israelii were found to increase with increased severity

of periodontal inflammation (Nisengard and Beutner, l970a).

By analysis of sera titers and use of F.A. tests,

Gilmour and Nisengard (1973} demonstrated serum antibody

activity against A. nae·slundii, ~ israelii, Bacteronema

matruchotti, and Lepto·trichia bucca.lis in patients with gin­

givitis and periodontitis.

The presence of local antibodies in inflamed human

gingiva has been documented (Hartzer, Toto, and Gargiulo,

1971, Byers, 1973, Marttala, Toto, and Gargiulo, 1974).

In a study of the cellular immune response in pa­

tients with periodontal disease, Chan, Baker, Mergenhagen,

and Socransky (1974) demonstrated the reaction of lympho­

cytes against ~ naeslundii, ~ viscos'us, A. israelii, and

s. mutans.

The antigenic activity of cell portions of ~ ~­

lundii has been demonstrated (Irving, Heeley, and Socransky,

1975). The authors found that intragingival injections of

whole dead cells, cell walls, or cytoplasm obtained from ~

naeslundii elicited an initial polymorphonuclear response at

34

the injection site in all cases. Destruction of osteoblasts

of adjacent alveolar bone was also observed for all cell

portions. Osteocyte destruction was noted subsequent to

whole dead cell or cytoplasm injections but not to cell wall

injections. Cell wall injections elicited an early appear­

ance of lymphocytes which "overshadowed" the polymorphonu­

clear response and persisted up to twenty-four hours post

injection, thus suggesting a delayed type hypersensitivity.

The destruction of osteoblasts and osteocytes seen after in­

jection of products of ~ naeslundii may explain in part

the bone loss seen in periodontal disease.

CHAPTER III

MATERIALS AND METHODS

Eight dark-eared, adult albino hamsters, four male

and four female, obtained from the Whitney Animal Labora-

tory, Aurora, N.Y., were used in this experiment. The ani-

mals were fed the Cariogenic Test Diet containing 61% su­

crose produced by Teklad Test Diets of Madison, Wisconsin.

The composition of this diet is given in Table III. All

hamsters were given the diet and water ad libit.um. The ani--

mals were divided into two groups: the control group which

consisted of two animals, one male and one female, and the

test. group, consisting of six animals, three males and three

females.

The six animals in the test group were exposed to

Actinomyces naeslundii* for a period of 71 days. Actinomy-

ces naeslundii is a gram-positive, p1eomorphic, branching, '

anaerobic rod adaptable to facultative growth (Socransky,

Hubersak, and Propas, 1970) and found in negligible numbers

in the oral cavity of the albino strain of hamster (Keyes

*obtained in lyophilized form from the American Type Culture Collection of Rockville, Maryland.

35

TABLE III

Composition of the Test Diet

CARIOGENIC TEST DIET

Alfalfa leaf meal • • •

Hi lk, po•ndered \\Thole

Sucrose • • • • •

Sodium Chloride •

Grn./Kg.

30.0

••• 350.0

• 610.0

. . . . . . 10.0

36

37

and Jordan, 19641. Exposure to this organism was accom­

plished by adding one ml. of a pure two-day culture of the

organism in trypticase soy broth. In addition, the test

animals were exposed directly by applicat~on of one ml. of

the two-day broth culture to the oral cavity of each ani­

mal via pipette. The oral administration was every three

days for the first three weeks, after which administration

was reduced to once every two weeks. The culture added to

the drinking water was replaced every five days with a

fresh, pure culture of A. n·aeslundii.

The A. naeslundii cultures administered -in the ex­

periment were two days old. The cultures were renewed by

transfer to fresh sterile trypticase soy broth every five

days and checked for purity by gram staining and micro­

scopic inspection.

For purposes of direct oral administration of the

test organisms, the experimental animals were anesthetized

with diethyl ether in a glass jar of three liter volume

(dessicator type) •

Application of the test organism consisted in place­

ment of two - four drops (0.1 - 0.2 ml) of culture to each

quadrant of the oral cavity. At each oral administration

of the microorganism the hamsters, both test and control,

were examined for signs of gingivitis. A positive sign

38

would consist in red, swollen gingiva as seen in the maxil­

lary and mandibular areas.

At the end of the experimental period of 71 days,

the blood from test and control animals was obtained. The

method of collection was as follows: test animals were

anesthetized with diethyl ether. The right brachial artery

of each was identified and transacted and the blood taken

up with a pipette. The blood from the test animals was

pooled in a sterile 10 ml. centrifuge tube. The same pro-

cedure was followed in obtaining the-blood of the control

group.

The next procedure was serum collection. The blood

in each tube (control and test) was centrifuged separately

for five minutes at 2000g in an International Equipment Com-

pany Model HN centrifuge. A pipette was then used to take

up the supernatant serum. This supernatant was then trans-

ferred to another sterile centrifuge tube and the process

repeated. These supernatant sera, test and control, were

then analyzed for the presence of serum antibodies specific

for the test antigen, A. naeslundii.

Two methods were used to determine the presence of

antibodies to A. naeslundii in the serum. Serial agglutina-

tion was used for rapid, less specific, identification. The

fluorescent antibody technique was employed for more specific

identification.

39

Antigen preparation:

The procedure used in antigen preparation was the same

for both the serial agglutination and fluorescent antibody

techniques. The supernatant trypticase soy broth was taken

up through a pipette, leaving an observable "clump" of A.

naeslundii cells at the bottom of the tube. To the tube

was added 5 ml. of O.l5N phosphate buffered saline solution

and the tube was again centrifuged for 3 minutes. The su­

pernatant saline was drawn off with a pipette and 5 ml. sa­

line added. This was centrifuged 3 minutes and the saline

drawrt off, leaving 0. 5 ml. to suspend ·tne bacter-ial cells.

1. Serial agglutination:

Clean microscope slides were prepared with

one drop of the microorganism suspended in the 0.15N saline

solution. To one drop of the organism in saline was added

one drop of serially diluted hamster serum. These dilu­

tions were: 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512,

1:1024. Agglutination was observed as clumping or aggregat­

ing of bacteria directly observed through the dark field

phase microscope.

2. Fluorescent antibody technique:

Microscope slides were prepared, placing one

drop of suspended microorganism on each slide. Two methods

were employed to fix bacteria to the slides: air drying and

40

heat fixation. One drop (0.05 ml.) of hamster serum was

then added to each slide. This procedure was followed in

preparing slides with both control and experimental sera,

five slides being made with each.

The slides were then incubated at 37qC for 35 min-

utes and carefully rinsed in phosphate buffered saline to

avoid dislodging the organisms. Then rabbit anti-hamster

IgG antiserum conjugated with fluorescein isothiocyanate

(FITC}* was applied to the slides and the incubation was

continued 35 minutes at 3~C. The slides were then rinsed

for three minutes with 0.15N phosphate buffered saline.

Cover slips were mounted on the slides using a drop of glyc-

erin. Observation for fluorescence of the bacteria was done

with fluorescent microscopy**. Photographs were made using

color film ASA 164, type B, Kodak***, at 30, 45, and 60

second exposures.

The hamsters were sacrificed by a lethal dose of

diethyl ether and a block of the mandible containing the

molar teeth of each animal, experimental and control, was

resected for histological study. The tissues were fixed in

10% formalin for 48 hours. They were then washed and dehy-

*Microbial Associates, Bethesda, Md., Cat. i 64-506 **Reichert Zetopan microscope using ultra-violet light

source HB200 American Optical, Rochester, N.Y. ***Kodak, Rochester, N.Y.

41

drated in ascendins alcohol concentrations and cleared in

xylene. They were then ·embedded in paraffin. ~ive repre­

sentative sections were cut at six microns with an A 0

Rotary 820 microtome from each block, and stained with hema­

toxylin and eosin.

CHAPTER IV

RESULTS

Fluorescent Antibodx Technique

Fluorescence of ~ naesl'undii, when treated with the

serum of experimental animals, was positive, demonstrating

the presence of antibodies specific for Actinomyces naeslun­

dii (See Figure 1 and 2). Fluorescence of A. naeslundii

when treated with serum from control animals was negative,

indicating the absence of such antibodues in the serum {See

Figure 3}. A table of results for experimental and control

groups is given in Table IV.

42

FIGURE 1: A thin smear of a suspension of Actinomyces

naeslundii cells in saline solution with ham­

ster sermn from experimental animals. This

serum had been treated with antihamster IgG

bound to fluorescein isothiocyanate (xlOO).

Note the fluorescence of the smear indicating

the presence of antibodies in the hamster

serum specific for A. naeslundii.

43

FIGURE 2: Dense smear of A. naeslundii cells in saline

with hamster antiserum from experimental ani­

mals treated with rabbit antihamster IgG im­

munoglobulin conjugated with FITC (x450).

Note positive fluorescence indicating the

presence of antibodies against ~ naeslundii

in the hamster serum (See Table IV) .

44

FIGURE 3:

45

Smear of A. naeslundii in saline with hamster

antiserum from control animals treated i n a

manner identical to that used for the experi­

mental serum: treatment with r abbit anti­

hamster IgG conjugated with FITC (x lOO). Note

negative fluorescence, indicating an absence

o f antibodies against ~ naeslundii in the

s erum of control animals (See Table IV) .

TABLE IV

Fluorescence of A. naeslundii reacted with

serum from experimental and control hamsters.

ANIMALS FLUORESCENCE

Control 1

Control 2

Experimental 1 +

Experimental 2 +

Experimental 3 +

Experimental 4 +

Experimental 5 +

Experimental 6 +

46

47

Agglutination

Rapid slide a~glutination was positive in control

animals, demonstrating a titer of 1:256 of antibody in the

serum sp-ecific for A. naeslundii. Agglutination was neg­

ative in control animals, indicating an absence of these

antibodies from the serum of these animals (See Table V) .

TABLE V

Agglutination of A. naeslu!ldii with serum

from control and experimental animals.

ANTIBODY TITER

Animal: 1:8 1:16 1:32 1:64 1:128 1:256 1:512 1:1024

Control 1

Control 2

Experimental 1 + + + + + +

Experimental 2 + + + + + +

Experimental 3 + + + + + +

Experimental 4 + + + + + +

Expe~imental 5 + + + + + +

Experimental 6 + + + + + +

48

Histopathology

Observation of histological slides of the mandibles

and maxillae of experimental and control animals demon­

strated the presence of a basophilic plaque of microorgan­

isms adjacent to the gingival sulcus epithelium in the in­

terproximal areas (See Figure 4}.

Signs of an incipient gingivitis were evident upon

observation of the histological sections in experimental

animals. The lamina propria and basement membrane showed

edema, dilated capillaries, and a few polymorph~nuclear

leukocytes. The PMN's had migrated to the epithelium

(See Figures 4, 5, 6, 7}.

In addition to edema and vasodilation, the lamina

propria showed a loss of collagen fibers. Further, the

alveolar bone in the crest area showed osteoclastic resorp­

tion (See Figures 4, 5, 8}.

Control animals showed normal gingivae with few

PMN's in the lamina propria. The alveolar crests of bone

showed smooth contour and the epithelium and connective

tissue appeared normal. (See Figures 9, 10, 11}.

49

FIGURE 4 : Bacterial plaque found on the gingi val sulcus

epithelium i n experimental -hamsters (x40). Note

the lamina propri a shows edema and dilated cap­

illaries, with a few PMN leukocytes indicative

of incipient gingivitis.

50

FIGURE 5: Early gingivitis in the hamster subsequent to

treatment with A. naeslundii (x lOO). Note the

edema of the basement membrane, dilated capil­

laries , and a few PMN leukocytes. There is a

dense fibrous lamina propria. The alveolar

crest shows oste oclastic resorption.

FIGURE 6:

51

Early gingivitis in the hamster showing the

interdental papilla of an experimental animal

(xlOO) . There is intercellular edema and PMN

leukocyte infiltration of the epithelium. The

lamina propria shows edema and loss of collagen

fibers with the infiltration of a few PMN leu­

kocytes.

52

FIGURE 7: High magnification of Figure 6 (x4 00). Note

PMN leukocytes and intercellular edema of gin­

gival epithelium.

53

FIGURE 8: Low magnification of Figure 5 (x25). Interden-

tal papilla in an experime~tal hamster showing

gingivitis and alveolar cres t bone resorption .

FIGURE 9:

54

Interdental papilla from a control hamster

(x40), showing normal epithelium and connective

tissue. Note few PMN's in the lamina propria

and presence of interproximal plaque.

55

FIGURE 10 : Hi gh magnification of F i gure 9 (x lOO). Note

few PMN's in the lamina propria and n ormal

epithelium .

56

FIGURE 11: Low magnification of Figure 9 (x25 ). Normal

interdental papilla from control hamster.

Note smooth contour of the alveolar crest and

normal epithelium and connective tissue.

CHAPTER V

DISCUSSION

This study has demonstrated the production of serum

antibodies di'rectly specific for· Actinomyces naeslundii by

albino hams'$!rs subsequent to an oral administration of this

microorganistl\ to these animals for a period of 71 days.

Histological·l sections taken from the molar areas of the max­

illae and ~hdibles of experimental animals showed the pres­

ence of interproximal plaque and an incipient gingivitis.

Interproximal plaque was observed in histological sections

taken fromthe control animals as well. The gingivitis seen

in the experimental animals was characterized by intercellu­

lar edema and polymorphonuclear leukocytic infiltration. The

lamina propria showed edema, loss of the basement membrane,

and slight infiltration with PMN leukocytes.

·It is probable that the constant challenge to the

test animals with A. naeslundii cells as antigens resulted

in an immunological response to this organism in these ham•

sters. The resulting antibody produced by the host could

then lead to the formation of antigen-antibody complexes.

These complexes are capable of initiating the complement

57

58

sequence, components of which can release chemical mediators

of inflammation (Snyderman, 1973}. In addition, these anti-

. gen-antibody complexes are directly chemotactic for polyn'tor­

phonuclear·leukocytes (Boyden, 1962}, the products of which

are known to contribute to tissue destruction (Fell, Coombs,

and Dingle, 1966, Cochrane, 1969).

r Althoughthere have been experiments in the past

which have inlplicated various strains of Actinomyces, includ­

ing ~ naesJtundii, as initiating factors of periodontal dis­

ease in thehamster, none has demonstrated the induced pro­

duction of serum antibodies specific for an organism which

was experimen~ally introduced into the model under study,

this being the main purpose of this investigation. Important

circumstances to be ·Considered are: Actinomyces species are

found.in negligible numbers in the normal oral flora of the

albino hamster, the model used in this study (Keyes and Jor"

dan, 1964): it has been demonstrated that this animal does

not develop periodontal disease when subjected to a high car­

bohydrate diet, unlike the cream and ;golden strains which do

develop the disease under similar circumstances {Keyes and

Jordan, 1964).

It has been demonstrated that bacterial plaque is

chemotactic for PMN's in vitro, the bacteria being the.pri­

mary chemotactic factors (Miller, Folke 1 and Umana, 1975).

59

Microscopic observation of the histological sections in this

study revealed the presence of a distinct plaque which had

formed interstitial to the molar teeth in control and exper­

imental animals. Also in evidence in experimental animals

was the presence of a few PMN's in the lamina propria, epi­

thelium, and adjacent to the plaque. This implies the pres­

ence of a chemotactic agent in the plaque of experimental

animals. It is possible that this agent consists of cells

or cell portions of A.· naeslundii administered to experimen­

tal animals. That certain strains of oral bacteria produce

a product di:rectly chemotactic for PMN' s is well documented

(Walker, Ba~let, and Kurtz, 1969, Tempel et al., 1970, ., --

Snyderman, 1973, among others) •

Wilderman (1962) demonstrated plaque components to

be nutrients for bacterial growth. The relationship of

plaque to gingival inflammation in experimental animals has

been studied at length (Saxe et al., 1967, Gad, 1968, Lindhe

et al., 1973, among others). That an-accumulation of bac­

terial plaque in man is directly correlated with increased

gingival inflammation has been shown (L8e, Theilade, and

Jensen, 1965) •

Gibbons and van Haute (1973) have made the observa-

tion that Actinomyces naeslundii forms plaque-like deposits

in culture. These cells produce surface constituents which

60

impart cohesive properties enabling them to form bacterial

masses. Actinomyces colonies form a filamentous "meshwork"

which can physically entrap other bacterial species. In

addition, certain species of Actinomyces are capable of

synthesizing polymers which bind dissimilar organisms. This

has been demonstrated by the use of the scanning electron

microscope (Jones, 1970}. A direct attachment between cells

was demonstrated in studies of thin sections by Listgarten,

Mayo, and Amsterdam (1973}.

~t is indeed probable that certain organisms are

able to acc~ulate in plaque only by means of in-terspecies

interactions because of a lack of alternate adherent mech­

anisms. The presence of significant numbers of these organ­

isms in plaque would require the proper interacting species

to be previously present on the tooth surface. This may be

the role of Actinomyces in plaque formation--that of the

interacting species. Bladen, Hageage, Pollock, and Harr

(1970) reported that Veillonella species were able to attach

to preformed plaques formed by ~ viscosus but that these

organisms could not adhere to hard surfaces in vitro. These

results may not be conclusive in view of the fact that these

hard surfaces were not apatite. Aggregation of cells of

Veillonella with suspensions of A. viscosus has been ob­

served (Gibbons and Nygaard, 1970}.

61

It may be by these mechanisms, of antigenicity and

plaque formation, that A.· na·e·s·lundii probably acts as a

primary etiological agent in the initiation of gingivitis

in albino hamsters (Jordan and Keyes, 1964, Slack, Mance,

and Gerencser, 1973, !rving, Heeley, and Socransky, 1975).

The incipient. gingivitis found in experimental ani­

mals in this investigation was similar to that reported by

Miller and Ripley (1975} in their study of early periodon­

tal disease ·in the Syrian hamster. The authors inoculated

experimental animals with A. nae·s lundii, using a method sim­

ilar to thai: of the present investigation. In addition, the

diet used, diet 2000, containing 56% sucrose, (Keyes and

Jordan, 1964) was similar to that of the present study.

Jordan and Keyes (1964} observed an induction of

gingivitis after a 71 day period in albino hamsters inocu­

lated with a labelled strain of Actinomyces. This strain

was recovered from the plaque of experimental animals upon

termination of the investigation. The development of an

incipient gingivitis in experimental hamsters after a 71

day test period in this investigation supports the findings

of their experiment. Diet 2000, used in their study and

similar to that used in this investigation, was successful

in supporting the implantation of this labelled strain of

Actinomyces in albino hamsters.

62

Not observed in this investigation were the changes

. grossly apparent in the periodontium of experimental ani­

mals reported by Socransky, Hubersak, and Propas (1970),

who experimentally induced periodontal disease in germfree

Sprague Dawley rats by a monoinfection with A. naeslundii.

One possible reason for the difference in severity between

the two experiments is the length of the test period. The

test period in this investigation was 71 days, while Socran­

sky et al. ran experiments ranging from 90 to 455 days. It

may be that the incipient nature of the disease seen in this

experiment was due to the relatively short test period. This

is plausible in view of the fact that the gingivitis in this

investigation was similar to that found by Miller and Rip­

ley (1975} in the Syrian hamster over a similar test period.

However, another possible reason for the incipient nature

of the disease seen in the test animals in this investigation

is the possibility that the repeated exposure to the test

antigen induced a sensitization in these animals, the anti­

body produced serving an immunologic function in defense of

the host and preventing a progression of the gingivitis.

The use of fluorescently labelled rabbit anti-ham­

ster IgG immunoglobulin, in "t::his experiment, proved to be

successful in the identification of antibodies specific for

A. naeslundii in the serum of albino hamsters previously

63

infected experimentally with this organism. The use of A.

naeslundii as an antigenic agent to challenge albino hamsters

was effective in inducing the production of serum antibodies

in the host specific for this organism, thus eliciting an

immune respbnse in these animals.

The 'fluorescent antibody technique is a highly re­

liable and -specific method of identification of antigen-

antibody complexes which are often not demonstrable by means

of agglutination or other types of serological tests {Cherry,

Goldman, and Carski, 1960). Fluorescent antibody techniques

have been used with consis~ent success in the identification

of bacterial antigens since 1953 {Goldman, 1953 and 1954).

The isothiocyanate derivative of fluorescein, the label

used in this investigation, has been effectively used in the

labelling of globulins since 1958 {Marshall, Eveland, and

Smith, 1958, Riggs, Seiwald, Burckhalter, Downs, and Met-

calf, 1958).

The successful employment of antiglobulins labelled

with FITC in the identification of A. naeslundii in this

study confirms the observations of Lambert, Brown, and

Georg (1967}. Significantly, the authors found A. naes----lundii to be serologically distinct from morphologically

similar A. israelii and demonstrated the fluorescent anti-

body test to be more specific than the agar-gel diffusion

64

test. The characterization of A.· n·aeslundii as serologi­

cally distinct was supported in 1970 by Socransky et al.

The albino hamsters in this study, both control

and experimental, were seen to develop an interproximal

plaque resulting from a high sucrose diet and, in experi­

mental animals, oral administration of A. naeslundii. It

is possible that the plaque of experimental animals con­

tained A. naeslundii cells, considering that such implan­

tation of Actinomyces in the plaque of albino hamsters has

been documented (Jordan and Keyes, 1964). A. naeslundii

has been specifically identified in dental plaque by the

use of fluorescent antibody (Snyder, Bullock, and Parker,

1967, Slack, Mance, and Gerencser, 1973).

In this investigation, a production of serum anti-

bodies in albino hamsters specific for ~ naeslundii was

demonstrated. This observation supports the findings of

Nisengard and Beutner (1970), Gilmour and Nisengard (1973), .

and Cummings and Hageage (1974), in human patients with

periodontal disease. In addition, this study strengthens

the observations of Michalek et al. (1976), who demonstrated

that the experimental ingestion of bacteria in gnotobiotic

rats results in a production of specific antibodies in

these animals.

65

This serum antibody production is a result of the

antigenic nature of the cells of Actinomy·ces· naeslundii.

The possible routes of entry of such antigens could be the

gingiva (Marttala, Toto, and Gargiulo, 1974} and the gastro­

intestinal tract as reported by Michalek et al. (1976).

The results of this investigation support the find-

ings of Irving, Heeley, and Socransky {1975}, who reported

antigenic activity on the part of cell portions of A. naes-

lundii.

Anaerobic culturing in trypticase soy broth provid--

ed for adequate growth of the bacterial cultures required

in this investigation. This was observed as rapid growth

to turbidity in the broth tubes. The water bottles of exper-

imental animals became turbid subsequent to the addition of

the pure broth culture of Actinomyces naeslundii. The water

bottles of the control group, however, remained clear. This

supports the observations of Socransky, Hubersak, and Propas

(1970) who characterized A. naeslundii as being adaptable

to facultative growth.

Rapid slide agglutination, using serum serially

diluted, proved to be a reliable method for the determina-

tion of a serum antibody titer. The dark field phase micro~

scope provided a useful means of observation of bacterial

clumping.

66 .

In preparation of ~A slides, heat fixation proved

to be superior to air drying in fixing the bacteria to the

slides.

This investigation points toward further studies

of the immunological mechanisms involved in antigen-host

interaction. In particular, further investigation of the

specific antigenic portions of bacterial cells and their

interaction with the host immune system is indicated. The

role of hypersensitivity in gingival disease, not yet fully

understood, must also be studied. Furthermore, the defen­

sive role of antibodies formed against plaque bacteria in

patients free of periodontal disease should be investigated.

CHAJ?TER VI

SUMMAR¥ AND CONCLUSIONS

Ac·tinomyces· ·naes·lundii from a broth culture was

prepared as a smear on slides and reacted with antiserum

obtained from six albino hamsters previously inoculated with

A. naeslundii and having. gingivitis. Smears were also pre­

pared with serum from two uninoculated albino hamsters hav­

ing normal gingivae. The treated smears were reacted with

rabbit antihamster antiserum to the immunoglobulin IgG con­

jugated with fl~orescein isothiocyanate. The slides were

then examined microscopically for immunofluorescence with a

fluorescent microscope with an ultraviolet light source.

Fluorescence was positive to Actinomyces naeslundii in the

serum of experimental animals and negative in the controls.

A. naeslundii from a broth culture was prepared as

a smear on slides and reacted with antiserum obtained from

the six experimental and two control animals serially di­

luted. The slides were observed through a dark field mi­

croscope for agglutination of the bacteria. Agglutination

was positive in the experiment~! animals with a serum titer

of 1:256 and negative in the controls.

67

68

A block of the mandibles and maxillae containing

the teeth of each animal was resected for histologic study.

Observation of slides made from these sections showed the

pres~nce of interproximal plaque in both control and experi­

mental animals. Signs of an incipient gingivitis were evi­

dent in experimental animals.

In •xperimental animals, the lamina propria and

basement·.snembrane showed edema, dilated capillaries, and a

few polymorphonuclear leukocytes. The PMN's had migrated

to the epithelium. The bone of the alveolar crest showed

osteoclastic resorption.

Heat fixation proved to be a better method than air

drying for fixing the bacteria to the slides. The bac­

terial cells showed better retention using heat fixation.

The following can be concluded from this study:

1. Animals treated with Actinomyces naeslundii via oral

inoculation, developed an incipient gingivitis.

2. The challenge of Actinomyces naeslundii induced the

production of serum antibodies specific for this

organism in experimental animals. Control animals

demonstrated no antibody production against ~ ~­

lundii.

3. Heat fixation is superior to air drying for purposes

of retaining the bacteria to the slide.

BIBLIOGRAPHY

Arnim, S.S. "Antony van Leeuwenhoek, the :F!rst Periodontist," Ac. Rev.·. ·of the Ac·ad. ·p·e·r·io., July 3, 1967. - - ......... -.--. ---.

Bahn, A.N. "Microbial Potential in the Etiology of Perio­dontal Disease, ••· J. Perio·dont., 41: 603-10, 19 70.

Baram, l?, andArnold, L. "Immunologic Aspects of Perio­dontal Disease," J. pe·rio"dont., 41: 617-19, 1970.

Beveridge and Goldner, M. "Statistical Relationship Be­tween the Presence of Human Subginsival Anaerobic Diphtheroids and Periodontal Disease, ... J.· Dent. Res., 52: 451-53, Jan.-June, 1973. - ---- ---

Bladen, Hageage, H.G., Pollock,F., and Harr, R. "Plaque Forma·tioo in Vitro on Wires by GraltJ,-Negative Oral Microorganisms (Veillonella} , " ~- ·~ Biol., 15: 127, 1970.

Blank, C.H. and Georg, L.K. "The Use of Fluorescent Anti­b()dy Me'thods for the Detection and Identification of Actinomyces Species in Clinical Material," J. Lab and Clin. Med., 71: 283-93, 1968. - ------

Boyden~ S.V. "The Chemotactic Effect of Mixtures of Anti­body and Antigen on Polymorphonuclear Leukocytes," J. Exp. Med., 115: 453, 1962.

Burnet7, G.W: and Scherp, H.W. Oral Microbiolof and Infec­t~ous D~sease: A Textbook ~Students an · Pract~ t~on­

. ers in Dent~str~7 3rd ed. Baltimcre: Williams and Wilkins Co., 1~ 8.

Byers, C.N. "Levels of the Immunoglobulins IgA, IgG, and IgM in the Inflamed Human Gingiva." Master's Thesis, Loyola University School of Dentistry, 64 pages, 1973.

Chan, S.P., Baker, J.J., Mergenhagen, S.E., and Socransky, s.s. "Lymphocyte Stimulation by Oral Bacterial Anti­gens," Abst. #329, J. ~· ~·, 53: 137, 1974.

69

70

Cherry, W.B., Goldman, Sc. D., and Carski, T.R. "Fluores­cent Antibody Techniques in the Diagnosis of Communi­cable Diseases," U.~. Dept. of Health, Ed., and Wel­fare, l?ublic Health Service, Bureau of.State Services Communicable Disease Center, Atlanta, Ga., Publication #729, 1960.

Cochrane, C.G. and Dixon, F.J. "Cell and Tissue Damage Through Antigen-Antibody Complexes," In Textbook of Immunopathology, pp. 94-100, 1969.

Cohn, Z.A. and Hirsch, J.G. "The Isolation and Properties of the Specific Cytoplasmic Granules of Rabbit Poly­morphonuclear Leukocytes," J. Exp. Med., 112: 983-1004, 1960. - ---

Coons, A.H., Creech, H.J., Jones, R.N., and Berliner, E. "The Demonstration of Pneumococcal Antigen in Tissues By the Use of Fluorescent Antibody," J. Immunol., 45: 159-1701 1942 • -

Cummings, N.A. and Hageage, C.J. "Antibodies to Actinomyces · viscosus in the Serum and Gingiva Extract of a Patient

With Severe Periodontal Inflammation," Abst. #330, J. Dent. ~., 53: 137, 1974.

Darwish, s., Hyyppa, T., Manganiello, A.D., and Socransky, S.S. "Predominance of Cultivable Microorganisms in Periodontitis and Periodontosis," II, Early Periodon­titis, Abst. #289, J. ~· Res., 52: 131, 1973.

Dick, D.S. and Shaw, J.H. "The Infectious and Transmissible Nature of the Periodontal Syndrome of the Rice Rat," ~· ~ Biol., 11: 1095-1108, 1966.

Dumonde, D.C., Howson, W.J., and Wolstencroft, R.A. "Fifth International Symposium on Immunopathology"(ed. R.A. Miescher, P. Grabar.) New York: Grune and.Stratton, Inc., p. 263, 1967.

Ellison, S.A. "Oral Bacteria and Periodontal Disease," J. Dent. Res., 49: 198-200, 1970.

71

Fell, H. B., Coombs, R.R., and Dingle, J .T. ''The Breakdown of Embryonic Cartilage and Bone Cultivated in the Pres­ence of Complement-Sufficient Anti-serum .•• ," ·Int. Arch. All~rgy ~-· Immunol., 30: 146, #2, 1966. --

Frenkel, J.K. pathology Society Symposium on Choice of Animal Models for the Study of Disease Processes in Man, Introduction, Fed. Proc., 28: 160-215, 1969.

Gad, T. and Langebaek, J. Paradontalbehandling af Furka­turinvolverede Taender, Tandlaegebladet 72: 331-40, April 1968 (Dan.l

Gibbons, R.J. and Nygaard, M. "Interbacterial Aggregation of Plaque Bacteria," Archs. Oral ~·, 15: · 1397, 19 70.

Gibbons, R.J. and Van Houte, J. "On the Formation of Dental Plaques,"!!· Periodont., 44: 347-60, 1973.

Gilmour, M.N. and Nisengard, R.J. "Sera Titers -to Filamen­tous Bacteria and Relation to Periodontal Disease," Abst. #333, J. ~· Res., 52: 142, 1973.

Goldman, M. "Cytochemical Differentiation of Endamoeba Histolytica and Endamoeba coli by Means of Fluores­cent Antibody,"·~· J. Hyg., 58: 319-28, 1953.

Goldman, M. "Use of Fluorescein-tagged Antibody to Identify Cultures of Endamoeba Histolytica and Endamoeba coli," ~- !!· ~., 59: 318-25, 1954.

Green, J.A., Cooperband, S.R., Rutstein, J.A., and Kibrick, S. "A Lymphocyte-produced Factor which Inhibits Pro­liferation of Other Cells," ~-·~., 29: 306, 1970.

Gupta, O.P., Auskaps, A.M., and Shaw, J .H. "Periodontal Disease in the Rice Rat IV: The Effects of Antibiotics on the Incidence of Periodontal Lesions," ~ Surg., 10: 1169-1175, 1957.

Hartzer, R.C., Toto, P.O., and Gargiulo, A.W. "Inunune Reac­tions in the Gingiva of the Pregnant and Nonpregnant Human Female," J. Periodont., 42: 239-45, 1971.

Haurowitz, F. Inununochemistry and the Biosxnthesis of Anti­bodies. N.Y., London, Sydney:--yofin Wiley and sons;-­P· 139, 1968.

72

Heidelberger, M. and Kabat, E.A. "Chemical Studies on Ag-. glutination," J. Exp. Med., 65; 885, 1937. ·

Helderman, W.H. van P. "Total Viable Count and Differential Count of Vibrio Sputorwn, Fusobacterium Nucleatum, Sel­enomonas Sputigena, Bacteroides Ochraceus, and Veillon­ella in the Inflamed and Non-inflamed Hmaan Gingival Crevice, .. !!· Periodontal Res., 10: 294-305, 1976.

Hill, J. and Ward, P.A. 11 C3 Leukotactic Factors Produced by a Tissue Protease, .. J.· E:xp. Med., 130: 505, Sept. 1, 1969. -

Hook, W.A., Snyderman, R., and Mergenhagen, S.E. "Histamine­Releasing Factor Generated by the Interaction of Endo­toxin With Hamster Serum," Infect. Immunity, 2: 462, Oct. 1, 1970.

• "Further Characterization of a Factor From ----~E~n-a-o-t~o-x~~-n-Treated Serum Which Releases Histamine and

Heparin from Mast Cells," Infect. Inununity, 5: 909, #6, 1972.

Horton, J.E., Leikin, S., and Oppenheim, J.J. "Human Lympho­proliferative Reaction to Saliva and Dental Plaque De­posits: An In-Vitro Correlation With Periodontal Dis­ease,"_i[_. Periodontal., 43: 522, Sept., 1972.

Horton, J.E., Oppenheim, J.J., and Mergenhagen, S.E. "Elaboration of Lymphotoxin by Cultured Human Peripheral Blood Leukocytes Stimulated With Dental Plaque Depos­its,'' ~· Exp. Inununol. # 13: 383, March 1, 1973.

Horton, J.E., Raisz, L.G., Simmons, a.A., Oppenheim, J.J., and Mergenhagen, S.E. "Bone Resorbing Activity in

· Supernatant Fluid from Cultured Hum~n Peripheral Blood Leukocytes, 11 Science, 177: 793, Sept. 1, 1972.

Irving, J.T., Heeley, J.D., and Socransky, s.s. "Cellular Response to Subgingival Injection of Bacterial Products in th~ Rat," J. Periodontal·~., 10: 324-31, 1975.

Ivanyi, L. and Lehner, T. "Stimulation of Lymphocyte Trans­formation by Bacterial Antigens in Patients with Perio­dontal Disease,"~· Oral Biol., 15: 1089, Nov., 1970.

73

Jensen, J. "Anaph.ylation in its Relation to the Complement System," Science, 155: 1122, March. 3, 1967.

Jensen, S.B., USe, H..E., Schiott, R., and Theilade, E. "Experimental Gingivitis in Man," IV, J.· Periodontal Res., 3: 284-93,· 1968. -

Jensen', S:.B. and Mergenhagen, S.E. "Influence of Endotoxins on the Dermal Response of Rabbits to Human Oral Bac­teria,"· Arch. Oral Biol., 9: 241-54, 1964.

Jensen, J.·; Snyderman, R., and Mergenhagen, S.E. Chemotac­tic Activity, a Property of Guinea Pig CS Anaphylatox­in, Proceedings of the Third International Symposium on Cellular and Humoral Mechanisms of Anaphylaxis and Allergy, Basel, Switzerland, Karger, p. 265, 1969.

Jones, S.J. "Natural Plaque on Tooth Surfaces: A Scanning Electron Microscopy Study," Apex, J.U. College Hosp., Dent. Soc., 5: 93, 1971

Jordan, H.V. "Rodent Model Systems in Periodontal Disease Research," J. ~- Res., 50: 236-42, 1971.

Jordan, H.V. and Keyes, P.H. "Aerobic, Filamentous Bacter­ia as Etiologic Agents of Experimental Periodontal Dis­ease in Hamsters," Arch. oral Biol., 9: 401-14, 1964.

Jordan, H.v., Keyes, P.H., and Bell.a.ck, s. "Periodontal Lesions in Hamsters and Gnotobiotic Rats Infected with Actinomyces of Human Origin," J. Periodontal Res., 7: 21: 1972. - -

Jordan, H.V., Keyes, P.H., and Lim, S'. "Plaque Formation and Implantation of Odontomyces Viscosus in Hamsters Fed Different Carbohydrate Diets," J. Dent. Res., 48: 824, 1969. - -

Keller, H.J. and Sorkin, E. "Studies on Chemotaxis," V, Int. Arch. Allergy~· Immunol., 31: 505, 1967.

Keyes, P.H. and Fitzgerald. "Dental Caries in the Syrian H.amster-x, 11 .!..!:!!· ~- J., 13: 86-109, 1970.

Keyes, P.H. and Jordan, H.V. "Periodontal Lesions in the Syrian Hamster-III," Arch. Oral Biol., 9:377-400, 1964.

74

King, J.D. and Gimson, _A.P. "Lesions of the Gum and Alveo­lar .. Sone," ar·it, J. ~·, 2: 111-18, 1948-49.

Klinkhamer, J.M. "Quantitative Evaluation of Gingival and Peri-odontal Di.sease.," p·eriodont., 6: 207-11·, 1968.

Krygier, G. 1 Genco, R.J., Mashino, P.A., and Hausmann. "Experimental Gingivitis in Macaca Speciosa Monkeys: Clinical, Bacteriologic, and Histological Similarities to Hllil\an Gingivitis.," J.· Periodontol., 44: 454, 1973.

Lambert, F.W., Brown, J.M., and Georg, L.K. "Identification of Actinomyces ·israeTii and A. naeslundii by Fluores­cent Antibody and Agar-gel DTffusion Techniques," J. Bacteriology, 94: 1287-95, 1967. -

Lindhe, J., Hamp, S.E., and Loe, H. "Experimental Periodon­titis in the Beagle Dog," J. Periodontal Res., 8: 1-10, 1973.

Listgarten, M.A., Mayo, H., and Amsterdam, M. "Ultrastruc­ture of the Attachment Device Between Coccal and Fila­mentous Microorganisms in 'corn cob' Formations of Den­tal Plaque," Archs. Oral Biol., 17: 651-56, 1973.

Loe, H., Theilade, E., and Jensen, S.B. "Experimental Gin­. givitis in Man," J. Periodontol., 36: 177-87, 1965.

Macdonald, J.B'., Gibbons, R.J., and Socransky, s.s. "Bac­terial Mechanisms in PeriodontalDisease," Ann. N.Y. Acad. Sci~, 85: 467, 1960. -- --

Macdonald, J.B., Socransky, s.s., and Sawyer, S.J. "Patho­.genicity Experiments with the Flpra of the Periodontium in· Rice Rats," J. ~· ~., 39: 861, 1960.

Macdonald, J.B., Socransky, S.S., and Sawyer, S.J. "Survey of the Bacterial Flora of the Periodontium in the Rice Rat,"· Archs. ~ Biol., 1: 1-7, 1959.

Marshall, J.D. Jr., Eveland, w.c., and Smith c.w. "Superi­ority of Fluorescein Isothiocyanate (Riggs) for Fluores­cent Antibody Technique with a Modification of its Ap­plication," Proc •. Soc. Exper. Bioi. & Med., 98:898-900, 1958. --

75

Marttala, W.H., Toto, l?.D., and Gargiulo, A.W. "Identifica­tion of Fluorescent Antibodies in Periodontitis," J. Periodontal., 45: 853-61, 1974.

Mergenhagen, S.E. "Nature and Significance of Somatic Anti­. gens of Oral Bacteria," J. bent. Res., 46: 46, 1967.

Michalek, S.M., Me Ghee, J.R., Mestecky, J., Arnold, R.R., and Bozzo, L. "Ingestion of Strepto·co·ccus mutans Induces Secretory Innnunoglobulin A and Caries Innnunity," Science, 192: 1238":"40, 1976.

Miller, R~L., Folke, L.E.A., and Umana, C.R. Ability of Dental Plaque Upon Autologous ous Human Polymorphonuclear Leukocytes, " tol.·, 46: 409-14, 1975.

"Chemotactic or Heterolog­J. Periodon-

Miller, W.A. and Ripley, J.F. "Early Periodontal Disease in the Syrian Hamster," J. Periodontal., 46: 368-74, 1975.

Moody, M.D., Goldman, M., and Thomason, B.M. "Staining Bac­terial Smears with Fluorescent Antibodies I. General Methods for Malleomyces pseudomalleii," J. Bact., 72: 357-61, 1956.

Moskow, B.S., Wasserman, B.H., and Rennert, M.C. "Spontan­eous Periodontal Disease in the Mongolian Gerbil," J. Periodontal. Res., 3: 69, 1968.·

Movius, D.L., Rogers, R.S. III, and Reeve, C.M. "Lympho­cytotoxicity for Gingival Epithelial Cells in Perio­dontal Disease," J. Periodontal., 46: 271-76, 1975.

Nisengard, R.J. and Beutner, E.H. "Immunologic Studies of Periodontal Disease V. IgG Type Antibodies and Skin Test Response to Actinomyces and.Mixed Oral Flora," J. Periodontal., 41:· 149-52, 1970a.

• "Relation of Immediate Hypersensitivity ----:t""":o:--"""';P;::"e:::oor~i-:::-o~d~on·ti tis in Animals and Man, " J. Periodontal. ,

41: 223-27, 1970b. -

Oppenheim, J.J. "Immunological Relevance of Antigen and Antigen-Antibody Complex Induced Lymphocyte Transforma­tion," Ann. Allergy, 27: 305, July, 1969.

76

Papermaster, D. "Immuno9lobulins, 11 In· 1~ono·gr·aph. ~ Immunol­~' p. 11, Kalamazoo, Mich.: The Upjohri Co., 1972.

Pick, E. and Turk, J.L. "The Biological Activities of Sol-uble Lymphocyte Products, 11 ~· ·Exp. ·Itn.nlunol., 10: 1, Jan., 1972.

Riggs, J.L., Seiwald, R.J., Burkhalter, J., Downs, C.M.,and · Metcalf, T.G. "Isothiocyanate Compounds as Fluores­

cent Labeling Agents for Inunune Serum,,,. Am. J. ·path., 34: 1081-97, 1958. - - --

Rizzo, A.A.· 11 Periodontal Disease Research, Introductory Remarks,''!!·~· Dent. Assoc., 87: 1019, 1973.

Rovin, s., Costich, E.M., and Gordon, H.A. "The Influence of Bacteria and Irritation on the Initiation of Perio­dontal Disease in Germ-free and Conventional Rats," J. Periodont. Res., 1: 193, 1966.

-Saxe, S.R., Greene, J.C., Bohannon, H.M., and Vermillion,

J.R. "Oral Debris, calculus, and Periodontal Disease in the Beagle Dog," Periodontics, 5: 217, 1967.

Schultz-Haudt, s., Bruce, M.A., and Bibby, B.G. "Bacterial Factors in Nonspecific Gingivitis," ~· Dent. ~., 32: 681, Oct., 1953, Abst. #117.

Schultz-Haudt, s., Dewar, M., and Bibby, B.G. "Effects of Hyaluronidase on Human Gingival Epithelium," Science, 117: 653, 1963.

Schwartz, J. and Diblee, M. "The Role of IgE in the Release of Histamine from Human Gingival ~ast Cells," J. Perio­dontal., 46: 171-77, 1975a.

• "The Effect of Endotoxins and Enzymes in Vitro ___ o_n_th,....-e-R·elease of Gingival Histamine," J. Periodontal.,

46: 662-68, 1975b. -

Seyle, H. The Mast Cells. Washington, D.C.: Butterworth and Co-.-, -19 6 5 •

Shaw, J.H., Griffiths, D., and Auskaps, A.M. "Influence of Antibiotics on the Periodontal Syndrome in the Rice Rat," J. ~- ~., 40: 511-19, 1961.

77

Slack, J .M., Lanfried, s., and Gerencs.er, .M.A. "Identifica­tion of Actinomyces and Related Bacteria in Dental Cal­culus by the Fluorescent Antibody Technique, •• J. ~· ~., SO: 78-82, 1971.

Slack, J.M., Mance, T., and Gerencser, M.A. "Fluorescent Antibody and Microscopic Counts of Filamentous Bacteria in Plaque," Abst. #155, J. ~· Res., 52: 96, 1973.

Snyder, M.L., Bullock, w.w., and Parker, R.B. "Morphology of Gram-Positive Filamentous Bacteria in Dental Plaque by Fluorescent Antibody Technique,". Ar·ch. Oral Biol., 12: 1269-73, 1967.

Snyderman, R. "Immunological Mechanisms of Periodontal Tis­sue Destruction," J. Amer. Dent. Assoc., 87: 1020-26, 1973. - ---- ----

Snyderman, R., Phillips, J.K., and Mergenhagen, S.E. "Bio­logical Activity of Complement in Vivo: Role of CS in the Accumulation of PMN's in Inflanunatory Exudates," J. Exp. Med., 134: 1131, 1971.

Snyderman, R., Shin, H.S., Phillips, J.K., Gewurz, H., and Mergenhagen, S.E. "A Neutrophil Chemotactic Factor Derived· from CS Upon Interaction of Guinea Pig Serum With Endotoxin," J. Inununol., 103: 413-22, 1969.

Socransky, s.s., Hubersak, C., and Propas, D. "Induction of Periodontal Destruction in GnotObiotic Rats by a Human Oral Strain of Actinomyces naeslundii," Arch. Oral Biol., 15: 993-95, 1970.

Socransky, s.s., Macdonald, J.B., Sawyer, and Auskaps, A.M. "Quantitative Studies of the Bacterial Flora of the Periodontium in Rice Rats," Arch. Oral Bio.l., 2: 104-10, 1960. - --

Socransky, S.S. and Manganiello, S.D. "The Oral Microbiota of Man From Birth to Senility," J. J?eriodont., 42: 485-94, 1971. -

Stetson, C.A. "Similarities in the Mechanism Determining the Arthus and Shwartzman Phenomena," J. Exp. Med., 94: 347-58, 1951.

78

Taichman, N.S., Friedman, H.L., and Uriukara, T. "Inflam­mation and Tissue Injury I. The Response to Intradermal Injections of Human Dento-Gingival Plaque in Normal and Leucopenic Rabbits,"~·· Or-al· B'iol., 11: 1385-92, 1966.

Tempel, T.R., Snyderman, R., Jordan, H.V., and Mergenhagen, S.E. "factors from Saliva and Oral Bacteria Chemotac­tic for Polymorphonuclear Leukocytes: Their Possible Role in Gingival Infla:rmnation," J •· ·pe·ri·odo·n·t·ol., 41: 71-80, 1970. -

Thilander, H. "The Effect of Leucocytic Activity on the Structure of the Gingival Epithelium in Man,"~· Odont. Scand., 21: 431-51, 1963.

Thomason, B.M., Moody, M.D., and Goldman, M. "Staining Bac­terial Smears with Fluorescent Antibodies II. Rapid De­tection of Varying Numbers of Malleomyces Pseudomallei in Contaminated Materials and Infected Animals," J. ~., 72: 362-67, 1956. -

Walker, W.S., Barlet, R.C., and Kurtz, H.M. "Isolation and Partial Characterization of a Staphylococcal Leucocyte Cytotaxin," J. Bact., 97: 1005-8, 1969.

Ward, P.A. "A Plasmin-Split Fragment of C3 as a New Chemo­tactic Factor," J. Exp. Med., 126: 189-206, 1967.

Ward, P.A., Lepow, I.H., and Newman, L.J. "Bacterial Fac­tors Chemotactic for Polymorphonuclear Leucocytes," ~· J. Path., 52: 725-36, 1968.

Ward, P.A. and Newman, L.J. "A Neutrophil Chemotactic Fac­tor from Human CS," J. Immunol., .102: 93-99, 1969.

Wilderman, J.D. "A Cellular Study of Plaque," ~· Progr., 2: 211, 1962.

Williams, B.L., Pantalone, R.M., and Sherris, J.C. "Subgin­gival Microflora and Periodontitis," J. Periodont. ~., 11: 1-18, 1976.

APPROVAL SHEET

The thesis submitted by John A. Ranieri has been read and approved by the following Corr~ittee:

Dr. Patrick D. Toto, Director Professor, Chairman, Departments of General and Oral Pathology, Loyola

Dr. Paul W. Goaz Professor, Vice Chairman, Oral Diagnosis, Loyola

Dr. Ioannis S. Scarpa Assistant Professor, Acting Chairman, Biochemistry, Loyola

The final copies have been examined by the director of the thesis and the signature which appears belo\'7 verifies the fact that any necessary changes have been incorporated and that the thesis is now given final approval by the Committee with reference to content and form.

The thesis is therefore accepted in partial fulfill­ment of the requirements for the degree of Master of Science.

:?¥,~ /f77 Date D1rector's Signature